Mitochondrial DNA sequence, karyotypic, and morphological variation in the Carollia castena species complex (Chiroptera: Phyllostomidae) with description of a new species

TEXAS TECH UNIVERSITY

Natural Science Research Laboratory

Occasional Papers

Museum of Texas Tech University

Number 254 26 June 2006

Mitochondrial DNA Sequence, Karyotypic, and Morphological Variation
in the Carollia castanea Species Complex (Chiroptera: Phyllostomidae)

with Description of a New Species

Editorial Comment. Texas Tech University (TTU) has constructed a new wing to the Natural Science Research Laboratory
of the Museum (cover photograph) that houses natural history collections. Such collections are expensive and labor intensive
to build and maintain. Is it a wise utilization of our resources to expand existing natural history collections? The answer
is complex because of an almost endless list of ways that such collections are valuable to society. Papers that discuss the
significance of natural history collections include: Yates, 1985, Acta Zoologica Fennica 170:81-82; Pettitt, 1991. Museum
Journal 91(8):25-28; Patterson, 2002, Mastozoologia Neotropical 9:253-262; Suarez and Tsutsui, 2004, Bioscience 54:66-
74; and Natural Sciences Collections Association, 2005, A Matter of Life and Death, Natural Science Collections, Why
Keep Them and Why Fund Them?, http://www.nhm.ac.uk/hosted_sites/natSCA/collections/AMatterOfLifeAndDeath.pdf.

Such collections serve as the foundation of understanding the biodiversity of life. What is it worth to know and appreciate
the diversity of life on earth? in your state? in your backyard? In addition to the joy of knowledge and the aesthetics of
understanding life, another value of natural history collections is identification of species of vertebrates and their parasites
that serve as reservoirs for diseases of humans and economically important animals. For example, collections at TTU and
the University of New Mexico served to resolve the origin of the so-called “Four Corners Hantavirus” that resulted in a
notable number of human deaths in 1993 (Yates et al. 2002. Bioscience 52:989-998). Tissue collections were critical to
establish that this disease was not generated by bio-warfare efforts; rather, a native mammal, the deer mouse ( Peromyscus
maniculatus ), is the natural host, and aerosol from feces and urine is the mode of transmission. This information permits
development of behaviors that reduce risk of catching this disease.

Another role for natural history collections is to document the body load of pollutants, such as radiation, that is present
in specimens as well as the biological consequences of bearing this load. The collection of mammals in the NSRL includes
over 3,000 specimens from the Chernobyl region of Ukraine that document the biodiversity in the world’s most radioactive
region, as well as the genetic profile of individuals present in the radioactive zone as compared to those living in the so-
called “clean” zones. TTU scientists, led by Dr. R. K. Chesser, have published over 25 papers using specimens from
Chernobyl to understand the significance of living in the Chernobyl environment. These Chernobyl specimens will be
available to scientists for future study.

Another value of natural history collections is that specimens can be used to design Ph.D. dissertations and master’s
theses, as well as research projects. The collections are literally a library of unread books about the story of life. Using
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centers of research. Institutions that have hired TTU graduates include: American Museum of Natural History, Arkansas
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Analysis and Synthesis (director), Oklahoma State University, Penn State University, Purdue University, Smithsonian
Institution, Texas A&M University, TTU, University of New Mexico, and Yale Medical School. In an overview, scientists
and educators in such positions serve society by generating basic knowledge that is used to make complex decisions that
are critical to society. The natural history collection at TTU is a tremendously valuable resource that makes students
competitive for excellent jobs and our faculty competitive for state, federal, and other grants that help achieve the mission of
the University. It is our goal to ensure that the quality of science and education justifies the existence of the natural history
collection at TTU.

Workings of the natural world, and man’s place in it, are mysteries that need attention. Epidemics, conservation, and
ecology are intertwined with the form and function of the earth’s organisms. We can neither protect ourselves from hazards
of nature nor benefit from its bounty without unraveling the complex linkages among the living species. Collections at
TTU are not just a depository of carcasses, but a cross section of real communities, and interacting taxa. We cannot gauge
change in our natural setting without reference of what it was before. We cannot predict where we are going without a
measure of how we have changed. The value of natural history museums accrues with time and will be coveted resources
in generations to come to serve as landmarks of what we were and what we are to become. Museums are often conceived
to preserve the past. But natural history museums are portals to our future. TTU thanks Ben E. Keith for this new wing,
and we will make every effort to wisely serve TTU and society with this resource.

RJB

Front cover: New wing of the Natural Science Research Lab from a southeast view. The date of publication of the
description of Carollia henkeithi is the date that the first catalogued specimens were transferred to the new wing. Funds
to build this wing were donated by Mr. Ben E. Keith. This new wing provides 136% increase in space for the collections.
Photo by Kathryn A. MacDonald.

Mitochondrial DNA Sequence, Karyotypic, and Morphological Variation
in the Carollia castanea Species Complex (Chiroptera: Phyllostomidae)

with Description of a New Species

Sergio Solari and Robert J. Baker

Abstract

Use of mitochondrial cytochrome-/? (cyt-Z?) gene sequences to address problems in bat
systematics has increased significantly in recent years. In the phyllostomid genus Carollia, it has
resulted in a more accurate taxonomy and a better understanding of genealogical relationships.
Thus far, eight species have been recognized, four of them described in the last five years by
using either morphology or a combination of morphology and DNA sequence data. Here, we
name and describe another new species, using a combination of cyt -b sequences, karyotypic data,
and discrete morphological characters. The new species is a member of the C. castanea species
complex, and until now it was included under that name. However, it possesses a distinctive
karyotype (2N=22, as opposed to 2N=20/21 in other species), its cyt -b sequence diverges by
almost 8.1% from that of C. castanea , it shows subtle but consistent differences in the upper teeth,
and it is smaller than C. castanea. Geographically, the new species is restricted to Peru, Bolivia,
and Brazil, south of the Amazon River, at elevations ranging from 200 to 1100 m. We discuss
the value of multiple datasets when recognition of a new species may be subject to debate.

Key words: Carollia castanea, cytochrome-/?, genetic species concept, karyotypes,
morphology, new species, systematics

Resumen

El uso de secuencias moleculares del gen citocromo-Z? para resolver problemas en
sistematica de murcielagos se ha incrementado significativamente en los ultimos anos. En el
genero de filostomido Carollia, esto ha permitido tener una taxonomia mas precisa y un mejor
entendimiento de las relaciones entre especies. A1 presente, ocho especies han sido reconocidas,
cuatro de ellas en los ultimos cinco anos, usando solamente morfologia o combinando morfologia
y analisis de datos moleculares. Nosotros describimos una nueva especie en base a secuencias
del citocromo-Z?, datos cariotipicos, y caracteres morfologicos discretos. Esta nueva especie
pertenece al complejo de especies de C. castanea , y hasta ahora habia sido incluida bajo ese
nombre; sin embargo, esta posee un cariotipo unico (2N=22, versus 2N=20/21 en otras especies),
su secuencia del citocromo-Z? diverge por casi 8.1% de C. castanea, muestra pequenas pero
consistentes diferencias en la denticion superior, y es comparativamente mas pequena que C,
castanea. La nueva especie se encuentra en Pern, Bolivia, y Brasil, al sur del Rio Amazonas, y su
rango altitudinal es entre 200 y 1100 m. Finalmente, discutimos el valor de multiples conjuntos
de datos cuando el reconocimiento de una nueva especie puede ser discutible.

Soi.ari and Baker, 2006. Mitochondrial DNA Sequence, Karyotypic, and Morphological Variation in the Carollia castanea Species

Complex (Chiroptera: Phyllostomidae) with Description of a New Species
Occasional Papers, Museum of Texas Tech University 254: i + 1-16

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Occasional Papers, Museum of Texas Tech University

Introduction

Research on systematic relationships among
Neotropical bats has been augmented in recent years
by the availability of modern methods. One method
which has shown utility in resolving phylogeographic
questions is DNA sequencing, with the mitochondrial
cytochrome-/? gene providing the most resolution thus
far (Avise 2000). The use of this approach for bats of
the genus Caro Ilia (Phyllostomidae) has provided a
more accurate taxonomy (Baker et al. 2002) as well as
clarified phylogenetic relationships among populations
(Hoffmann and Baker 2003).

Although only four species ( brevicauda , castcmea ,
perspicillata , and subrufa) were included in the last
account of Carollia (Koopman 1993), eight species
are currently recognized, including colomhiana
Cuartas et al. 2001, sowelli Baker et al. 2002, manu
Pacheco et al. 2004, and monohemandezi Munoz
et al. 2004. Carollia sowelli was first identified as
a consistent and divergent clade in an analysis of
molecular (mitochondrial DNA) sequences (Wright et
al. 1999). It is unlikely that C. sowelli could have been
distinguished from C. breviemdaa therwke, because no
significant morphological features were recognized to
set them apart (Pine 1972; Owen et al. 1984), although
McLellan (1984) found consistent size differences
between northern and southern populations of what
was then regarded as C. brevicauda . Further analyses
and increased sampling justified the description of C.
sowelli (Baker et al. 2002).

Molecular phylogeography was also used to
further study geographic variation among populations
of C castanea. Previously, Patton and Gardner (1971)
had identified a karyotypic race (2N=22) in specimens
from southeastern Peru, which departed from the
2N=20[9]/21 [(?] found in populations of Costa Rica and
Colombia (Baker and Bleier 1971), and eastern Ecuador
(Li in and Engstrom 1998). Using morphometric
variation, McLellan (1984) suggested a size cline in this
species too, with smaller individuals in the southern part
of the range (central Peru) as compared to individuals
from Central America. The phylogeographic analyses
by Hoffmann and Baker (2003) supported the hypothesis
that geographic groups in C. castanea might represent
more than one evolutionary lineage, by showing them
as clades divergent from each other by >7% (Kimura-2
parameter; Kimura 1980).

Although no morphological distinction had been
previously recognized in C castanea , the situation
was similar to that in other pairs of cryptic species,
such as C. brevicauda and C. sowelli (Baker et al.
2002), Rhogeessa tumida and R. ge noway si (Baker
1984), or Notiosorex crawfordi and /V. cockrumi
(Baker et al. 2003). Given this level of genetic and
karyotypic variation and their implications for species
boundaries, we describe a new species based primarily
on chromosomal and DNA sequence evidence. We
provide a morphological diagnosis for the species of
bats previously recognized as C. castanea and assess
the observed morphological variation in the context of
molecular and karyotypic variation.

Materials and Methods

We examined morphological and morphometric
traits of the geographic populations used in the
molecular analyses by Hoffmann and Baker (2003).
To determine the extent of variation in these traits, we
enhanced the taxonomic and geographic sampling by
including specimens from other museum collections.
However, this study does not represent a complete
revision of this group of species. We also included
four specimens used in the original report of the
2N=22 karyotype, to make sure they correspond with

our taxonomic decisions. Only adult animals (based
on fusion of epiphyses of metacarpals and phalanges;
Pine 1972) were used in taking measurements and
ascertaining diagnostic characters. Specimens
examined are listed in the Appendix.

Five geographic units were defined based on the
phylogenetic analyses presented by Hoffmann and
Baker (2003). Two units correspond to the groups 1
(eastern Ecuador) and 2 (Peru and Bolivia), two are

Solari and Baker-Variation in the Carollia castanea Species Complex

3

formed from group 3 (western Ecuador, and Costa
Rica plus Honduras and Panama), and the final one
(Colombia and Venezuela) was not represented in the
tree of Hoffmann and Baker (2003). External and
cranial discrete characters used in previous studies
(Hahn 1907; Pine 1972; Owen et al. 1984; Pacheco et
al. 2004) were recorded for each geographic unit.

External measurements were taken from skin
tags. Forearm length (FA) was measured on dry skins
or fluid-preserved specimens. In addition, the following
skull dimensions (to the nearest 0.01 mm) were taken:
greatest skull length (GSL); condyloincisive length
(CIL); postorbital width (POW); greatest width of
braincase (BRW); palatal length (PL); breadth across
canines (CC); maxillary toothrow length (MXTR);
breadth across the outer edges of the second upper
molars (M2M2); dentary length (DL); and, mandibular
toothrow length (MDTR) (Table 1). Measurements
were subjected to a multivariate analysis (MANOVA)
to test the null hypothesis that no significant differences
exist among the vector of means for geographic and
taxonomic groups. Differences were considered
significant for P < 0.05.

Molecular data were obtained following
Hoffmann and Baker (2003). We sequenced the
cytochrome-/? gene of four additional individuals from
Peru (see Appendix); these sequences were compared
with others available at GenBank or produced by our
lab, including those of the outgroups ( Glyphonycteris
sy/vestris and Trlnycteris nicefori ; see Baker et al.
2003) and representatives of other recognized species.
Phylogenetic relationships were estimated using the
neighbor-joining algorithm (Saitou and Nei 1987),
using Kimura-2 parameter distances (Kimura 1980) as
implemented in PAUP4.0 blO (Swofford 1999).

Shorter sequences (100-200 bp) were obtained
from skin clips for four specimens (MVZ 136460,
136462-4) with karyotypic data but no tissue samples,
to verify they matched the longer sequences of other
specimens representing the new species. A specific
protocol and designed primers were developed to
obtain these sequences (M. C. Knapp and K. Nelson,
in prep.).

Taxonomic History

Although no synonyms are known for Carollia
castanea , the distribution attributed to the species
increased as animals similar to the Central American
ones were discovered in South America (see below).
The current known distribution of C. castanea (sensu
lato) extends from Honduras into Peru, Bolivia, western
Brazil, and Venezuela (Koopman 1993). Allen (1890)
described the species from a single specimen with type
locality in Costa Rica; its distribution was extended to
Panama (Goldman 1920), Ecuador and Peru (Thomas
1920), and then to Honduras and Guyana (Goodwin
1942). Hershkovitz (1949) recorded castanea from
northern Colombia, Cabrera (1958) listed it as present
in Guyana, Colombia, Ecuador and Peru, and Husson
(1962) recorded it from Suriname. In French Guiana,
Brosset and Dubost (1967) also assigned some bats
to castanea. Shortly later. Pine (1972) updated the
distribution for all the known species in the genus,
including records of C. castanea from Bolivia.

Compounding the problem, the name castanea
was employed for the smaller species of Carollia
in South America, sometimes including what is
now called brevicauda , but also used as conspecific
with subrufa in Middle America (Felten 1956; Hall
and Kelson 1959). Records from Ecuador and
Peru (Thomas 1920) were later re-identified as C.
brevicauda by Tuttle (1970) and Pine (1972). Although
Hershkovitz (1949) discussed the taxonomy of most
other bats in northern Colombia, he provided no
further information on castanea. Judging from the
data presented by Husson (1962) from Suriname, his
castanea appears to be brevicauda (Genoways and
Williams 1979). Pine (1972) confirmed the separation
of C. castanea from C. brevicauda , and stated that
the specimen from Guyana (in Goodwin 1942) was
similar to but not conspecific with C. castanea , calling
it Carollia sp.? (1). Records from French Guiana were
re-identified as C. brevicauda by Brosset and Charles-
Dominique (1990).

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Occasional Papers, Museum of Texas Tech University

The first indication of genetic variation within
Camilla was reported by Patton and Gardner (1971);
karyotypes of Peruvian C. castanea (sensu lato) were
shown to be distinct from those of other Carollia
species, which have, typically, a sex-chromosome
system with diploid numbers of 20 in females (XX)
and 21 in males (XYjY,; Hsu et al. 1968). Patton
and Gardner (1971) regarded that karyotype as the
ancestral condition for Carollia , because it showed the
basic sex-chromosome system, with 2N=22 for both
males and females. The typical system (2N=20-21)
has been found in specimens of C. castanea (sensu
lato) from Costa Rica and Colombia (Baker and Bleier
1971) and eastern Ecuador (Lim and Engstrom 1998; D.
Parish, unpubl. data). Hsu et al. (1968) showed that the
small Yj attaches end-to-end to the short arm of the X
chromosome, and the long acrocentric Y 7 synapses with

the long arm of the X. Thus, the long arm of the X is
homologous to Y v Based on G-banding patterns, Stock
{1975) confirmed the X autosome relationship proposed
by Hsu et al. (1968) of the extra sex-chromosomes in
specimens from Costa Rica and Colombia and that the
extra Y was an autosome translocated without a sex¬
determining role. Carollia is a classical example of an
X-autosome translocation.

Following, we provide a morphological diagnosis
for the Carollia castanea clade of Fig. 3 of Hoffmann
and Baker (2003), and describe a new species based
on specimens from eastern Peru and Bolivia. We
compare this new species with populations from Middle
America, which would bear the name C. castanea in
its restricted sense.

Results

Morphological diagnosis of Carollia castanea
(sensu lato)

Carollia castanea (sensu lato) includes the
smallest and the most divergent members of the genus.
Several external, cranial, and dental traits are common
to all: fur color usually chestnut, but varying from
dull, dark gray-brown to pale tan; banding ill-defined,
but darkest at tips; forearm short and naked, its length
usually less than 38 mm; uropatagial notch shallow;
skull small and delicate; greatest skull length (GSL)
< 21 mm; maxillary toothrow (MXTR) < 6.9 mm;
braincase globular with well-developed anteorbital
processes and low sagittal crest; second upper
premolar (P4) considerably lingual to the labial
edge of the first upper molar, creating a prominent
notch in the outline of the toothrow' (Pine
1972); upper canines slender and elongated;
upper external incisors greatly reduced,
peg-likeor spicule-like; anterior cingular style of P4
greatly reduced or short and never in contact with P3;
anterior portion of the bony palate concave, the posterior
projection long and narrow, and the mesopterygoid fossa is
v-shaped; outer lower incisors rather reduced but not
concealed dorsally by cingula of canines (Pine 1972;
Koopman 1994); first lower premolar (p2) noticeably
lower than the second (Pine 1972); crown of first lower

molar (ml) extremely low; mid-internal (metaconid)
cusp of the third lower molar reduced to undeveloped;
and coronoid process of the mandible low, relative to
the canine height.

Emended diagnosis of Carollia castanea
H. Allen 1890

The following synonymy includes most of the
relevant taxonomic and distributional works, but it does
not constitute a full synonymy.

Carollia castanea H. Allen 1890: 19. Type locality:
[Angostura,] Costa Rica

[Hemiderma] castaneum : Elliot 1904: 670. Name
combination.

Hemiderma castaneum: Hahn 1907: 116
Carollia castanea: Miller 1924: 54
C.[arollia] castanea: Felten 1956: 199 (part)

Carollia castanea castanea: Hall and Kelson 1959:
125 (part)

Carollia castanea: Pine 1972: 17 (part)

Carollia castanea: Koopman 1993: 186 (part)

From the examination of the holotype of Carollia
castanea (USNM 36384) and other specimens from
Costa Rica and Honduras, this is clearly one of the most

So lari and Baker-Variation in the Carollia castanea Species Complex

5

divergent species of the genus. The following traits can
be considered diagnostic for this species. Dorsal color
varying from dull, dark gray-brown or chestnut to pale
tan; tricolor fur banding not well-defined. Forearm
and legs appearing naked, with only sparse small
hairs. Skull small and delicate with well-developed
anteorbital processes. Narrow contact between the
cingula of the upper canine and first premolar. The
second upper premolar (P4) is displaced lingually
with respect to the first upper molar. There is a short
and blunt anterior projection of the cingulum of P4
toward P3; P4 in close contact with M1, through a short
anterior projection of the cingulum of M1 that contacts
P4. Lower premolars graded, the first being smaller
rather than subequal; there is an evident gap between
these teeth. Measurements of the holotype (from Costa
Rica), and Central and South American specimens are
presented in Table 1.

Diploid chromosome number (2N) = 20-21,
fundamental number (FN) = 36, with an autosome
translocated to the subtelocentric X chromosome
(Baker and Bleier 1971; Stock 1975), which shows a
marked secondary constriction on the longer arms.

As restricted here, this species is distributed from
Flonduras (Goodwin 1942) to Panama (Goldman 1920)
in Central America, and in northern South America
from southwestern Venezuela (Handley 1976) into
Colombia (Hershkovitz 1949) and western Ecuador
(Albuja 1999).

Although the need for recognition of additional
entities at the species level may be open to question,
we consider that under both a phylogenetic (Cracraft
1983) and a genetic species concept (Dobzhansky
1950), each of the clades in the cytochrome-/? gene tree
is sufficiently divergent to be considered valid species
(see Bradley and Baker 2001). In addition, there are
cranial and dental characteristics allowing these groups
to be recognizable morphologically. Thus far, we have
no evidence of sympatry; however, we do not expect
distributional overlap given that the distributions are
probably delimited by major geographic features, such
as the Andes or the Amazon River (Hoffmann and
Baker 2003; Pacheco et al. 2004),

A new species of Carollia
Gray 1838

Carollia benkeithi, new species
Carollia castanea'. Pine 1972 (part)

Carollia castanea : Koopman 1978 (part)

Carollia castanea : Koopman 1993 (part)

Carollia castanea : Pacheco et al. 1995 (part)

Carollia castanea : Fonseca et al. 1996
Carollia castanea : Anderson 1997

Holotype .-An adult female deposited at the
Natural Science Research Laboratory (NSRL) of the
Museum of Texas Tech University (TTU 46187),
caught by a field team including Robert J. Baker,
Jane A. Groen (field number JAG 3549), Robert D.
Owen, Michael J. Smolen, and Priscilla K. Tucker, on
13 October 1983 at 2 km S of Tingo Maria, Province
of Leoncio Prado, Department of Huanuco, Peru,
at approximately 9°18’S, 75°59’W (Stephens and
Traylor 1983). The holotype consists of a skin and
skull, both in good condition, plus frozen tissues (TK
22892). External measurements (in millimeters):
total length 65; tail length 12; hind foot 11; ear 17;
forearm (dry) 33.68. Weight was not recorded. Cranial
measurements (in millimeters): greatest length of the
skull 18.99; condyloincisive length 17.44; postorbital
width 5.46; greatest width of braincase 9.09; palatal
length 8.01; breadth across canines 4.29; maxillary
toothrow length 6.05; breadth across outer edges of
second upper molars 6.66; dentary length 12.48;
mandibular toothrow length 6.56 (Table 1).

Distribution.-We have examined voucher
specimens of Carollia benkeithi from the lowland
forests of eastern Peru (Departments of Cusco,
Huanuco, Junin, Madre de Dios, and Ucayali), and
northwestern Bolivia (Departments of Beni and La Paz)
(see Appendix). Additional reports, under the name
C. castanea , have been provided by Anderson (1997),
Ascorra et al. (1993), Eisenberg and Redford (1998),
Koopman (1978), Pacheco et al. (1993), Patterson
(1992), Pine (1972), Tuttle (1970), and Uieda (1980).
All of these records reveal the range illustrated in
Figure 1, with the elevational range from 200 to 1100
m (Patterson et al. 1996). However, the true extent of
the species’ range is not known at present, even with
the large existing collections from South America.

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Occasional Papers, Museum of Texas Tech University

Table 1. Selected measurements (as defined in the text) ofC arollia benkeithi and C. castanea, including the holotypes
(TTU46J87 and USNM36384, respectively). All measurements are in millimeters. Summary statistics (mean ± stan¬
dard deviation [above], observed range, and sample size [below]) are provided.

Holotype

TTU

46187

C. benkeithi

Holotype

USNM

36384

C. castanea

HBL

65

60.85 ± 4.32

60.94 ± 4.31

52.0-68.0(27)

51.0-67.0 (39)

TL

12

9.26 ±2.18

8

8.94 ±2.07

5.0-14.0(27)

5.0-14.0(38)

HF

11

10.67 4t 1.30

10

11.19'% 1.24

8.0-14.0(27)

8.0-14.0 (40)

E

17

16.93 4 1.82

15

17.35 ±. 1.23

11.0-20.0 (27)

15.0-20.0 (38)

FA

33.68

35.70 ±0.70

%

35.670 ± 0.92

33.68-37.21 (29)

34.24-37.13 (31)

GSL

18.99

19.27 ± 0.30

19.98

19.60 ± 0.44

18.70-19.94 (31)

18.98-20.94(48)

CIL

17.44

17.45 ±0.28

17.72

17.71 ±0.42

16.89-18.01 (30)

16.70-18.76(48)

POW

5.46

5.33 ±0.17

5.10

5.43 ± 0.17

5.06-5.79 (31)

5.10-5.83 (48)

BRW

9.09

8.84 ± 0.18

8.94

8.96 ±0.21

8.39-9.24 (31)

8.47-9.33 (48)

PL

8.01

8.02 ±0.18

8.03

8.27 ±0.38

7.62-8.35 (31)

7.07-9.14 (48)

MXTR

6.05

6.07 ±0.12

6.28

6.25 ±0.22

5.74-6.29 (31)

5.82-6.91 (48)

M2M2

6.66

6.78 ±0.20

6.53

6.73 ±0.21

6.38-7.08 (31)

6.27-7.14(48)

CC

4.29

4.34 ±0.14

4.19

4.35 ±0.16

4.06-4.59 (29)

3.94-4.74 (46)

DL

12.48

12.57 ± 0.23

12.97

12.82 ± 0.39

11.97-13.06 (31)

12.43-13.89 (48)

MDTR

6.56

6.65 ± 0.15

6.20

6.81 4 0.23

6.41-6.93 (31)

6.39-7.36 (47)

Morphological diagnosis . - Carollia benkeithi
is a small Carollia with chestnut to dull-gray
brown dorsal fur; short and naked forearm, a
tuft of hairs at the base of thumb; legs short and
apparently naked; uropatagium wide, with shallow
distal notch. Skull relatively broad; a low sagittal
crest in some individuals; rostrum slender; high
forehead; interorbital constriction well-defined, making
the anteorbital region appear inflated; braincase
globular (Fig. 2). Second upper premolar (P4) displaced
toward the lingual side of the toothrow, making a
break in the lateral outline of the toothrow; however,
this tooth is not in close contact with the first molar

(Ml) and there is no projection of the cingulum of Ml
toward P4. A robust anterior projection of the cingulum
of P4 extends toward P3 (Fig. 2). A small, reduced
gap is between the bases of the lower canine and first
premolar. The space between the lower premolars is
reduced, but they are never in close contact. Cusps of
the first lower molar reduced, almost inconspicuous
in side view. A small accessory cusp on the postero-
lingual side of the third lower molar (m3) is always
present. The angular process of the mandible is short
and stout. Measurements of additional specimens of
C. benkeithi are included in Table 1; their localities are
listed in the Appendix.

Solari and Baker-Variation in the Carollia castanea Species Complex

7

Figure 1. Distribution of the three species of the Carollia castanea species complex. Localities for C. benkeithi , new
species, as determined by specimens examined and literature references. The stars represent type localities for C. benkeithi
and C castanea. Some localities are lumped for purposes of graphic representation.

8

Occasional Papers, Museum of Texas Tech University

Figure 2. Ventral view of the skull and mandible of the holotype of Camilla benkeithi (TTU 46187) and
a specimen of C. castanea (TTU 13177) from Honduras.

Solari and Baker-Variation in the Carollia castanea Species Complex

9

Karyotypic diagnosis. -Diploid chromosome
number (2N) = 22 in both males and females,
Fundamental number (FN) = 38; there is a single
pair of medium-small acrocentrics in the autosomal
complement (which are translocated to the X
chromosome in other Carollia species; Patton and
Gardner 1971), and the X chromosome is a small
submetacentric (Fig. 3). Carollia benkeithi also lacks
much of the heterochromatin in comparison to C.
brevicauda and C perspicillata (Stock 1975).

Molecular diagnosis 1 1 1 0 bp of the
mitochondrial cytochrome-/? gene from 4 additional
specimens of Carollia , assigned to C. benkeithi ,
based on morphology and distribution, were obtained.
These have been deposited at GenBank [DQ 177279-
177282], Another 4 sequences available from GenBank
(AF 512002-004 [Bolivia], AF187021 [Peru]), also
represent C. benkeithi. The Neighbor Joining tree
using Kimura-2 parameter distances for these and other
species of Carollia is shown in Fig. 4. The average
distance value that separates C. benkeithi from C.
castanea (sensu stricto) is 8.1%, and ranges from 7.3
to 9.1%. Genetic differences within the two clades in
C. benkeithi averages 1.7%.

A third clade emerging from this tree includes
two samples from eastern Ecuador, which diverge
from C. castanea by 8.3% and from C. benkeithi by
8.1 %. Presently, we recognize this genetically defined
phylogroup as a third unnamed species of the C.
castanea species complex (the C. castanea of Koopman
1993), although we do not provide a name for it. Based
on a preliminary revision of voucher specimens, this
species has a diploid chromosome number (2N) = 20-
21 (Lim and Engstrom 1998; D. Parish, unpubl. data),
and is distributed in eastern Ecuador (Albuja 1999) and
northeastern Peru (Pirlot 1968).

Codon position changes that distinguish C.
benkeithi from C. castanea include 27 fixed changes in
3 rd position, one in 2 nd position, and one in 1 sl position,
involving 25 transitions and 4 transversions. One
transition (position 917; T in castanea , C in benkeithi)
and one transversion (position 925; T/C in castanea ,
A in benkeithi ) resulted in two fixed amino acid
replacements.

Shorter sequences (ranging from 80-150 bp)
were obtained from the individuals with karyotypic
data (MVZ 136460, 136462-4) and compared to
the available sequences using the neighbor-joining
algorithm of PAUP. The four sequences clustered
together with the specimens representing C. benkeithi ,
thus confirming the observed morphological similarity
among the voucher specimens. These sequences are
available from the authors on request.

Description. -A small species of Carollia , with
long, fluffy fur on back. Dorsal pelage without sharply
defined banding; a broad buffy-chestnut band at the
base, followed by a brown-yellowish band, and then
narrow chestnut to dull gray-brown tips. Ventral pelage
with short bicolored, brown-tipped hairs throughout.
Forearm short (< 38 mm) and apparently naked; short
legs, apparently naked. The uropatagium with a
shallow and rounded notch.

Skull delicate, but relatively broad; a low sagittal
crest sometimes present; rostrum slender, with a high
forehead. Interorbital constriction well-defined,
making the anteorbital region appear inflated; braincase
globular. Posterior extension of the palate shorter
than anterior portion. Maxillary roots delicate and
usually presenting a pointed labial margin, oriented
dorsally. One or two small spines on the antero-
internal wall of the bullae; when two spines are
present, they are connected by a low ridge at their
bases. Angular process of the mandible are short
and stout. Elongated and slender upper canines,
slightly projected forward. Outer upper incisors
spicule-like, much smaller than the middle ones.
Second upper premolar (P4) displaced toward
the lingual side of the toothrow, producing a
break in the lateral outline of the toothrow. and with
a robust anterior projection toward P3. Anterior
cingulum of the first upper molar (Ml) does not project
toward P4. Lower incisors subequal in size, their
occlusal outline slightly convex. A small, reduced gap
between the bases of the lower canine and first premolar
(p2). Second lower premolar (p3) almost twice as high
as the first lower molar (ml), the cusps of which are
reduced and inconspicuous in side view. Third lower
molar (m3) proportionally small, with a small accessory-
cusp on the postero-lingual side. Mandibular rami and
toothrows almost straight.

10

Occasional Papers, Museum of Texas Tech University

H

Eft jlAift IIUX!

M

Figure 3. Karyotype of a female specimen of 'Camilla henkeithi , MVZ 136462 (courtesy of James L, Patton).

Comparisons., • Wc provide comparisons of
Carollia henkeithi with samples of C. castanea ,
within which it usually has been included (Pine 1972;
Koopman 1993). In the absence of a more accurate
understanding of the degree of variation within and
among populations of C. castanea , we assume that
populations from Costa Rica, where the type locality is
located, are representatives of that species. However,
a full review of the variation in this group of species
is beyond the objectives of this study. When more
voucher specimens are accompanied by sequence and
chromosomal data, such analysis will provide the most
powerful resolution.

Using size alone, Carol l ia henkeithi is readily
separable from the larger species of the genus, as
well as by means of several pelage and cramo-dental
features (Pine 1972; Pacheco et al. 2004). Camilla
henkeithi is most similar in all respects to C. castanea:
both species have variations of chestnut or pale brown
pelage, apparently naked forearms, and a shallow notch
in the edge of the uropatagium. In fact, C. henkeithi
is hardly distinguishable by external characters from
C. castanea , and their eranio-dental characters are
also similar. A few dental characters are useful in
differentiating C. henkeithi from C. castanea , including

the following: (a) a robust projection of the anterior
cingulum of P4 toward P3 in henkeithi { Fig. 2), which
is reduced to a short and blunt projection in castanea ;

(b) P4 not in contact with Ml in henkeithi (Fig. 2), or
if so then there is no development of a projection of
the anterior cingulum of M1; in castanea , an anterior
cingular projection of Ml makes contact with P4; and

(c) a small cusp on the postero-lingual side of m3 in
henkeithi (Fig 2), which is rarely present in castanea.

McLellan’s (1984) analyses provided early
evidence of geographic differences within Carollia
castanea (sensu lato). Although all of her 22
variables showed the lowest means for C. castanea as
opposed to other species of Carollia, she found that
locality variation within castanea accounted for
29.25% of the total variation. Five measurements
showed overall variation over 40%; three of these
were related to skull length, and two to skull width.
This variation corresponded with latitude, because
her samples from Peru (which came from close to the
type locality of C. henkeithi ) had the smallest values
in all but two measurements, and five of them showed
significant differences between what we recognize as
C. henkeithi and C. castanea.

Solari and Baker-Variation in the Carollia castanea Species Complex

11

G. sylvestris

T. nicefori

j -FN 37061 E Ecuador

~L FN 37065 E Ecuador
r NK 30033 Bolivia
NK 30150 Bolivia

I NK 25385 Bolivia
TK 125125 Peru
TK 125124 Peru
TK 22892 Peru
TK 70672 Peru

i 1

FN 38166 Panama
TK 38156 Panama
r FN 44029 C. Rica
TK 101378 Honduras
L FN 44016 C. Rica
r TK 104508 W Ecuador
■TK 104681 W Ecuador

■c

&

L- TK 104506 W Ecuador
r- FN 38212 Panama
> FN 38195 Panama
TK 46010 brevicauda
TK 104530 brevicauda

_r TK 104613 perspicillata

I FN 31809 perspicillata
jTK 101341 sowelli
™1-TK 101013 sowelli

I- TK 19550 subrufa

"I-TK15818 subrufa

CD

<D

C

42

to

CD

O

Figure 4. Phylogenetic relationships among seven species of Carollia as indicated by the neighbor joining tree based on
Kimura-2 parameter distances. Outgroups are Glyphonycteris sylvestris and Trinycteris nicefori.

benkeithi

12

Occasional Papers, Museum of Texas Tech University

Our data also show significant differences in most
of the eraniodental measurements in both MAN OVA
tests. When the five geographic groups were compared,
the differences among the vectors of means were
statistically significant (Wilks’ Lambda = 0.177; df =
50,290.7; P < 0.001), and all but one variable (M2M2)
showed significant differences. A second MANOVA
test including only two taxonomic entities (C. castanea
and C. benkeithi) also found significant differences
(Wilks’ Lambda - 0.623; df = 10, 59; P < 0.002), but
in this case two measurements (CC and M2M2) showed
no differences between the groups.

Remarks. The genus Carol Ha includes species
that are common to abundant almost everywhere in
the Neotropics (Pine 1972), and C. benkeithi is not an
exception. Based on our records and several others
(as C. castanea ), this species is typically found in
tropical evergreen forests at lower elevations, mostly
below 1000 m. Based on their overall resemblance,
the ecological and reproductive habits of C. benkeithi
should be similar to those of C. castanea.

Etymology. -The specific epithet benkeithi is
a modified Latin genitive after Mr. Ben E. Keith, a
long-time benefactor of the Natural Science Research
Laboratory (NSRL) of the Museum of Texas Tech
University. Species such as Carollia are not only
hard to tell apart, but often deemed too common to
merit specific focus of systematic studies. Funding
of research institutions has a direct impact on our
work and allows for significant effort on many poorly
understood taxa. A recent grant by Mr. Keith and
his family has resulted in a new wing that more than
doubled the size of the NSRL and greatly improved the
available facilities. We acknowledge his commitment
to the study of natural science collections by naming
this new species after him. Date of publication of this
new name was chosen to coincide with the day the first
catalogued mammal specimens were transferred to the
museum wing constructed through Mr. Ben Keith’s
generosity.

Discussion

An inability to discriminate between cryptic
species may lead to serious underestimation of
biodiversity, the perception of misleading biogeographic
patterns, and misinterpretation of ecological data
(Dorbigny et al. 2003). These misperceptions may
be critical in the case of health-related issues, such as
rabies, Bolivian hemorrhagic fever, etc., where proper
recognition of host species is vital. However, access
to sufficient biological information that would allow
indisputable recognition between congeneric species is
frequently unavailable. Rather, we depend on particular
sets of characters to shape a useful and convincing
species concept (Bradley and Baker 2001).

Evidence from nucleotide variation ot the
cytochrome-6 gene (Hoffmann and Baker 2003) and the
karyotypic polymorphism involving sex-chromosomes
(Baker and Bleier 1971; Patton and Gardner 1971)
concerning what was previously recognized as a
single species C. castanea (Koopman 1993) has
convinced us that the proper action was to recognize
this taxon as a species complex. Molecular sequence
divergence among phyllostomid bats is indicative ol
specific distinction at values between 5-7% (Bradley

and Baker 2001). In the order Chiroptera there are few
chromosomal races or chromosomal polymorphisms
(Baker 1979). The sex chromosome translocation
discussed in this paper is most parsimoniously regarded
as occurring at the base ot the radiation ol Cufollia
and may be a synapomorphy for the remainder of the
genus after C. benkeithi diverged from the common
ancestor for the genus. Cytochrome-6 data (Hoffmann
and Baker 2003) suggest that either C. castanea (sensu
lato) is paraphyletic and the translocation to the X
has occurred twice or, alternatively, there has been a
reversal to the primitive character state in C. benkeithi
(see also Lim and Engstrom 1998).

Pine (1972) discussed the diversification of
CarollkL concluding that castanea (sensu lato) would
be the most distinctive species in the genus; and
Me Lei lan (1984) stated that C. castanea was the most
morphologically distinct species in the genus. The
same conclusion has been reached using molecular
data (Lim and Engstrom 1998; Wright et al. 1999;
Hoffmann and Baker 2003), but phylogenies based on
morphological characters are missing.

Solari and Baker-Variation in the Carollia castanea Species Complex

13

The morphological and morphometric information
provided by the analyses of representative samples of
C. castanea (sensu lato) has allowed us to support
the results of Hoffmann and Baker (2003) regarding
the recognition of unidentified species of Carollia. A
similar approach has proven useful previously (Baker et
al. 2002) and we hope to complement the morphological
description of C. manu (Pacheco et al. 2004) with
karyotypic information and mtDNA sequences. Thus,

we could refine our current hypotheses on the origin
and diversification of this widespread genus, that
now includes 10 species: C. perspicillata (Linnaeus
1758), C. brevicauda (Schinz 1821), C. castanea H.
Allen 1890, C. subrufa (Hahn 1905), C. colombiana
Cuartas et al. 2001, C. so we Hi Baker et al. 2002, C,
manu Pacheco et al. 2004, C. monohernandezi Munoz
et al. 2004, C. benkeithi Solari and Baker 2005, and
one unnamed from eastern Ecuador and Peru.

Acknowledgments

Our appreciation goes to James L. Patton who
kindly provided two unpublished karyotypes of C.
benkeithi , one of which is presented in our Fig. 3. Federico
Hoffmann (ULN) produced most of the molecular data
and helped us with many of the phylogeographic
analyses. Michelle C. Knapp and Kimberlyn Nelson
developed the protocol and Steven R. Hoofer and K.
Nelson, the primers to obtain cyt -b sequences from
skin clips of specimens of C. benkeithi. Deidre Parish
karyotyped a specimen of Carollia sp. (TTU 84903)
from eastern Ecuador. Jorge Salazar (TTU) and the
writer’s guild of BIOL6100 (Fall 2004), Ronald
H. Pine, and F. G. Hoffmann made significant
contributions to previous versions of this manuscript.
Hugo Mantilla (TTU) created the map of Fig. 1 using
ArcMap 8.x in ArcGIS Desktop (ESRI) and produced
the digital images of Fig. 2. James E. Sowell in 2001,
and J. E. Sowell and Alan D. Brown in 2004 funded
the Sowell Expeditions (NSRL) that supported our
field work at Ecuador and Honduras, whereas National
Science Foundation (DEB 9870191) and the Marshall
Field Fund of the Field Museum funded field work

at Manu Biosphere Reserve. An Ernst Mayr Travel
Grant in Animal Systematics, from the Museum of
Comparative Zoology, Harvard University, and a
scholarship from the J. Knox Jones, Jr. Memorial Fund,
Texas Tech University, supported the visit of SS to the
USNM to study the holotype of C. castanea and other
Carollia specimens from Peru. We thank the curators
and other staff of the museums that allowed us to use
the collections under their care: Bruce D. Patterson and
William Stanley (FMNH), William L. Gannon and Terry
L. Yates (MSB), Victor Pacheco (MUSM, Lima, Peru),
J. L. Patton and Carla Cicero (MVZ), Heath Garner
(NSRL-TTU), Mark D. Engstrom and Burton K. Lim
(ROM, Ontario, Canada), and Don E. Wilson, Alfred L.
Gardner, and Linda Gordon (USNM). We also received
permission for use of tissues stored in these institutions.
Ron Chesser and Robert Bradley assisted in developing
the ideas in the Editorial Comments. Publication and
research activities supported by Texas Tech University
Fund for the Biological Database Studies.

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A ddresses of authors:

Sergio Solari

Department of Biological Sciences and

Natural Science Research Laboratory, The Museum

Texas Tech University

Lubbock TX 79409-3131 USA

e-mail: sergio. solari@ttu. edit

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Brasileira (Chiroptera, Phyllostomidae). Acta Amazonica
10:936-938.

Wright, A. J., R. A. Van Den Bussche, B. K. Lim, M. D. Engstrom,
and R. J. Baker. 1999. Systematics of the genera Carollia
and Rhinophylla based on the cytochrome-6 gene. Journal
of Mammalogy 80:1202-1213.

Robert J. Baker

Department of Biological Sciences and

Natural Science Research Laboratory, The Museum

Texas Tech University

Lubbock TX 79409-3131 USA

e-mail: robert. baker@ttu. edu

16

Occasional Papers, Museum of Texas Tech University

Appendix

List of specimens examined and their geographic localities. Specific localities are abbreviated to the major
geographic reference, based on the collectors’ labels. When two numbers identify a specimen the first one is the
museum catalog number, and the second is the tissue number [FN for ROM, NK for MSB, and TK for TTU].
Acronyms for museums and institutions follow Hafner et al. (1997).

Camilla castanea (Total: 51). HONDURAS:
Atlantida , Lancetilla (3: TTU 84037 [TK 101378],
TTU 84121 [TK101462], TTU 84386); Comayagua,
Cueva de Taulabe (1: TTU 84037); Olancho , 12.1 mi
by road SSW of Dulce Nombre de Culmi (3: TTU
13176-77, TTU 28100). COSTARICA: Heredia , 7.3
mi SE Puerto Viejo (3: TTU 13184-85. TTU 13487),
Parque Nacional Braulio Carrillo (1: USNM 562812);
Union, Estacion Biologica Cano Palma (1: ROM
108303 [FN 44029]), Tortuga Lodge (1: ROM 108291
[FN 44016]); San Jose , Angostura (1, Holotype:
USNM 36384). PANAMA: Chiriqui, Ojo de Agua
(1: ROM 104305 [FN 38156]), Santa Clara (1: ROM
104315 [FN 38166]); Darien , Parque Nacional Darien
(2: ROM 104341 [FN 38195], ROM 104353 [FN
38212]); San Bias, Armila (4: USNM 335199-200,
USNM 335204-05); Veraguas , lsla Cebaco (2: USNM
360170-71). COLOMBIA: Antioquia, Zaragoza (4:
USNM 499323-24, USNM 499326-27); Valle , Rio
Zabaletas (2: USNM 483411-12). VENEZUELA:
Tachira, 45 km NE San Cristobal (2: USNM 419508,
USNM 419510); Territorio Federal Amazonas, 32 km
SSE Puerto Ayacucho (4: USNM 407893-95, USNM
407897). ECUADOR: Esmeraldas, 7 km N Quimnde
on Quininde-Esmeraldas highway (1: USNM 522165),
San Lorenzo (3: TTU 85278 [TK 104506], TTU 85280
[TK 104508], TTU 85453 [TK 104681]); Guayas,
Balao, 10 km ESE Huerta Negra (2: USNM 498858,
USNM 522164); Pichincha, Santo Domingo, Rio
Palenque Science Center (8: USNM 528503-10).

Carollia benkeithi (Total: 47). PERU: Cusco,
La Convencion, Camisea (11: MUSM 13564, MUSM
13567, MUSM 13573, MUSM 13577, USNM 577783
[TK 70672], USNM 582800, USNM 582805-09);
Huanuco , Leoncio Prado, Tingo Maria, 2 km S (1,
Holotype: TTU 46187 [TK 22892]); Junto. 3.2 km N
Vitoc, Rio Tulumayo (5: USNM 507179-83); Madre
de Dios , Albergue Maskoitania, Rio Alto Madre
de Dios (4: FMNH 174603 [TK 125124], FMNH
174605 [TK 125125], FMNH 174607, FMNH 174609
[TK 125127]), Manu, Pakitza (12: MUSM 6837-41,

USNM 564376-78; USNM 566511-14); Ucayali,
Balta, Rio Curanja (4: MVZ 136440, MVZ 136462-4).
BOLIVIA: Beni, Yacuma (1: MSB 68356 [NK 25385]);
Cochabamba, Sajta (1: MSB 70297 [NK 30150]), Villa
Tunari (1: MSB 70298 [NK 30033]); La Paz, 1 mi W
Puerto Linares (1: TTU 34814-20).

Carollia unnamed species (Total: 9). ECUADOR:
Napa, Parque Nacional Yasuni (2: ROM 103979 [FN
37061], ROM 103983 [FN 37065]; Pastaza, Amazonas
Military Fort (1: TTU 84903), Taculin, below Puyo (2:
USNM 548109-10), Tiguino, 130 km S of Coca (2:
USNM 574522-23); Zamora-Chinchipe. Cumbartza, 3
km NE (1: USNM 513443). Los Encuentros, 4 km ENE
(1: USNM 513444). PERU: Loreto, Puerto Indiana
(Pirlot 1968).

Carollia brevicauda. ECUADOR: Esmeraldas,
San Lorenzo (TTU 85302 [TK 104530]); PERU:
Loreto , Quebrada Aguas Negras (MUSM uncataloged
[TK 46010]).

Carolliaperspicillata. ECUADOR: Esmeraldas,
San Lorenzo (TTU 85385 [TK 104613]); GUATEMALA:
El Peten , Poptun (ROM 99259 [FN 3 1809]).

Carollia sowelli. HONDURAS: Comayagua,
Cueva de Taulabe (TTU 82495 [TK 101341]),
Francisco Morazan, Parque Nacional La Tigra (TTU
82497 [TK 1010131).

Carollia subrufa. EL SALVADOR: Ahitachapan,
El Refugio (ROM 35506 [TK 15818]); MEXICO:
Jalisco, C ha me la (TTU 37719 [TK 19550]).

Glyphonycteris sylvestris. GUYANA: Siparuni,
Iwokrama Reserve (ROM 107445 [TK 16374]).

Trinycteris nicefori, VENEZUELA: Guarico, 45
km S Calabozo (Universidad Central de Venezuela,
UCV [TK 15189]).

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Library of Congress Cataloging-in-Publication Data
Occasional Papers, Number 254
Series Editor: Robert J. Baker

Mitochondrial DNA Sequence, Karyotypic and Morphological Variation in the Carolua castanea
Species Complex (Chiroptera: Phyllostomidae) with Description of a New Species
By: Sergio Solari and Robert J. Baker

ISSN 0149- 175X

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